Potential cancer-related role of circadian gene TIMELESS suggested by expression profiling and in vitro analyses

The circadian clock and cell cycle are two global regulatory systems that have pervasive effects on the behavior and physiology of eukaryotic cells. The 24-hour periodicity of the circadian rhythm, consisting of light and dark phases which coincide with the phases of the solar day, is maintained by a set of core circadian genes through a complex mechanism involving transcription-translational feedback loops [1, 2]. The cell cycle is monitored by a sequence of molecular and biochemical events including a series of checkpoint mechanisms to ensure completion of biochemical reactions unique to each phase of the cell cycle prior to initiation of subsequent phases [3, 4].

While these two regulatory systems involve distinct mechanisms, there is evidence that they are linked and interact at the gene, protein, and biochemical levels [5, 6]. A recent study has indicated that one circadian regulator, TIMELESS, is also a core component of the cell cycle checkpoint system [7]. It regulates directly or indirectly the activity of autoregulatory components of the mammalian circadian core, including Clock, Per, and Cry proteins, associates with S phase replication checkpoint proteins Claspin and Tipin, and is required for the phosphorylation and activation of Chk1 by ATR and ATM-dependent Chk2-mediated signaling of DNA double strand breaks [8, 9].

Although the connection between cancer and the cell cycle machinery that controls cell proliferation has been evident for some time, and there is mounting evidence to suggest that disruption of the circadian rhythm may increase susceptibility to certain malignancies [10‐12], little is known about TIMELESS’s role in tumorigenesis. Our previous case–control study demonstrated significant genetic and epigenetic associations of TIMELESS and breast cancer risk [13]. A recent study has also shown that higher levels of TIMELESS expression in colorectal cancer tissue is associated with TNM stages III-IV and microsatellite instability [14]. In contrast, findings from another study point to the down-regulation of TIMELESS in hepatocellular carcinomas [15].

In the current study, we report our findings from the expression profiling analysis of TIMELESS in different tumor types using publically available online tools and microarray datasets, and a loss-of-function analysis using TIMELESS-targeting siRNA oligos followed by a whole-genome expression microarray and network analysis. We also tested one of the potential roles of TIMELESS suggested by our network analysis using a MTS assay and observed that TIMELESS knockdown decreased the proliferation rate of MCF7 breast cancer cells.

Since the hypothesis linking circadian disruption to increased breast cancer risk was first proposed twenty years ago, there have been many molecular epidemiologic studies implicating the tumorigenic importance of circadian variations, including genetic and epigenetic variations, and aberrant gene expression [10, 57, 58]. TIMELESS, which regulates directly or indirectly the activity of autoregulatory components of the mammalian circadian core, has been shown to play an essential role in the cell cycle checkpoint response [8, 9]. As a potential molecular bridge between the cell cycle and the circadian regulatory systems, TIMELESS is also likely to play a significant role in tumorigenesis.

In our previous breast cancer case–control study, we found significant associations between two tagging SNPs in the TIMELESS gene and decreased breast cancer susceptibility. TIMELESS promoter hypomethylation in peripheral blood lymphocytes was also found to be significantly associated with later-stage breast cancer. In the current study, we observed that TIMELESS is frequently overexpressed in tumor relative to normal tissues in several cancer types, and that elevated expression of TIMELESS is significantly associated with later tumor stages and poorer breast cancer prognosis. Our findings also provide the first evidence suggesting the diagnostic and prognostic potential of TIMELESS in cancer.

Intriguingly, all 26 genes in the top IPA-generated network have been reported to be involved in cancer. G0S2 (3.37-fold increase), which encodes a mitochondrial protein that specifically interacts with Bcl-2, is a proapoptotic factor, and its ectopic expression induces apoptosis in diverse human cancer cell lines in which endogenous G0S2 is normally epigenetically silenced [48]. Similarly, RhoB (2.16-fold increase) is a well-characterized small GTPase that can inhibit cell proliferation, survival and invasion, and it is often down-regulated in cancer cells [47]. EMP1 (5.33-fold increase) encodes a potential tumor suppressor that is associated with cellular proliferation and metastasis [49]. DMBT1 (Deleted in malignant brain tumors 1 protein) (5.26-fold decrease) is a putative tumor suppressor gene frequently deleted in brain, gastrointestinal and lung cancers and down-regulated in breast cancer and prostate cancer [59]. Interestingly, Superoxide dismutase (SOD2), a probable tumor suppressor responsible for the destruction of superoxide free radicals [44], displayed a 15.9-fold increase in expression following TIMELESS knockdown. Additionally, Endothelin-1 (EDN1) (4.26-fold increase) encodes a growth factor that is frequently produced by cancer cells and plays a key role in cell growth, differentiation, apoptosis, and tumorigenesis [27]. Bone Morphogenetic protein 7 (BMP7) (2.41-fold increase), also known as osteogenic protein 1 (OP-1), encodes a multifunctional growth factor belonging to the TGF-β superfamily. Elevated BMP7 levels are reported to be correlated with the depth of colorectal tumor invasion, liver metastasis and cancer-related death [60], as well as the levels of estrogen and progesterone receptor, both of which are important markers for breast cancer prognosis and therapy [61]. Similarly, GDF15 (4.49-fold increase), which encodes another member of the TGF-β superfamily, was reported to exert proapoptotic and anti-tumorigenic functions on colorectal, prostate, and breast cancer cells in vitro and on colon and blioblastoma tumors in vivo[62]. IL8 (5.1-fold increase) has also been reported to have functions in the regulation of angiogenesis, cell growth and survival, leukocyte infiltration, and modification of immune responses [63]. These data suggest that loss of TIMELESS expression has the potential to influence a set of cancer-relevant genes, although most of these genes showing altered expression may not interact directly with TIMELESS. However, without further mechanistic investigations, it is not possible to identify whether these transcripts are direct or indirect targets of TIMELESS.

Timeless, together with its constitutive binding partner, Tipin, functions as a replisome-associated protein which interacts with components of the endogenous replication fork complex [64]. Moreover, siRNA-mediated TIMELESS down-regulation attenuates DNA replication efficiency [64]. Consistent with this observation, we observed a significant decrease in MCF7 cell proliferation after TIMELESS knockdown. However, we found only a slight but non-significant decrease in cell proliferation in HeLa cells following TIMELESS knockdown. This latter observation is consistent with the finding that TIMELESS down-regulation did not have a significant effect on cell proliferation in HeLa cells previously reported by Masai et al. [65]. As a recent study conducted by Engelen et al. revealed elevated TIMELESS expression in tissues undergoing active proliferation, the implication is that increased TIMELESS expression may be a characteristic of all highly proliferative cells, rather than one exclusive to cancer tissues. However, this relationship does not necessarily diminish the significance of TIMELESS in cancer simply because heightened cellular proliferation can be an important driver of the cancerous state. Even if TIMELESS expression is elevated as a result of, rather than a precursor to, heightened proliferation, TIMELESS expression may represent a natural response to abnormal proliferative rates and its potential physiological significance in cancer cannot be discounted. Further mechanistic studies are needed to investigate the precise role of TIMELESS on cellular growth and proliferation in different cancer types, as well as the capacity of TIMELESS to influence other potentially cancer-relevant pathways, including cell motility, invasiveness, and DNA damage response.

Although initial screening found a similar anti-proliferative response to a second siRNA, only the siRNA that conferred the greater phenotypic effect was chosen for subsequent assays. Given the inherent difficulty in controlling for off-target effects in any knockdown experiment performed using a single siRNA, the results presented here should be subjected to independent validation with use of a second siRNA. Furthermore, there is evidence to suggest that the anti-proliferative response observed from TIMELESS silencing could be partly attributable to apoptosis. It is evident that proliferation of transfected cells plateaus between the 48 hour and 72 hour time points and decreases thereafter, marking a period of gradual cell death. The degree to which silencing of TIMELESS elicits an apoptotic response should be the subject of a future investigation.

In summary, these findings, although preliminary, support the findings from our previous breast cancer case–control study, and provide further evidence of the link between TIMELESS and carcinogenesis. The expression profiling analysis of the tissue-specific microarray data suggests that TIMELESS is frequently overexpressed in various types of tumor tissues, and elevated TIMELESS expression is associated with advanced tumor stage and poorer breast cancer prognosis. These data, in conjunction with the findings from the network analysis and the cell proliferation assay, suggest TIMELESS may be involved in the tumorigenic process. However, further mechanistic investigations are warranted to further elucidate the precise role of TIMELESS in tumorigenesis, and to help in the development of targeted therapeutic strategies.